Changes in diffusible indole-3-acetic acid from various parts of tulip plant during rapid elongation of the flower stalk

In order to chemically identify the putative indole-3-acetic acid (IAA) and to confirm the native source of auxins account for rapid elongation of the floral stalk of tulip, we examined diffusible IAA from various parts of tulip plant during rapid elongation of the flower stalk. IAA was identified in the diffusates collected from the leaves, internodes, and floral organs with gas chromatography (GC)–mass spectrometry. The amount of diffusible IAA from different plant organs followed the order of that the internodes > flower organs > leaves during the period of rapid elongation of the floral stalk. The diffusible IAA from internodes reached its peak amount at different time than did diffusible IAA from the flower. The results obtained indicated that the top internode is probably the major source of auxins account for rapid elongation of the flower stalk.     

Characteristics of the digestive vacuole membrane of the alga-bearing ciliate Paramecium bursaria

Cells of the ciliate Paramecium bursaria harbor symbiotic Chlorella spp. in their cytoplasm. To establish endosymbiosis with alga-free P. bursaria, symbiotic algae must leave the digestive vacuole (DV) to appear in the cytoplasm by budding of the DV membrane. This budding was induced not only by intact algae but also by boiled or fixed algae. However, this budding was not induced when food bacteria or India ink were ingested into the DVs. These results raise the possibility that P. bursaria can recognize sizes of the contents in the DVs. To elucidate this possibility, microbeads with various diameters were mixed with alga-free P. bursaria and traced their fate. Microbeads with 0.20μm diameter did not induce budding of the DVs. Microbeads with 0.80μm diameter produced DVs of 5-10μm diameter at 3min after mixing; then the DVs fragmented and became vacuoles of 2-5μm diameter until 3h after mixing. Each microbead with a diameter larger than 3.00μm induced budding similarly to symbiotic Chlorella. These observations reveal that induction of DV budding depends on the size of the contents in the DVs. Dynasore, a dynamin inhibitor, greatly inhibited DV budding, suggesting that dynamin might be involved in DV budding.     

Structure of a human telomeric DNA sequence stabilized by 8-bromoguanosine substitutions, as determined by NMR in a K+ solution

The structure of human telomeric DNA is controversial; it depends upon the sequence contexts and the methodologies used to determine it. The solution structure in the presence of K(+) is particularly interesting, but the structure is yet to be elucidated, due to possible conformational heterogeneity. Here, a unique strategy is applied to stabilize one such structure in a K(+) solution by substituting guanosines with 8-bromoguanosines at proper positions. The resulting spectra are cleaner and led to determination of the structure at a high atomic resolution. This demonstrates that the application of 8-bromoguanosine is a powerful tool to overcome the difficulty of nucleic acid structure determination arising from conformational heterogeneity. The obtained structure is a mixed-parallel/antiparallel quadruplex. The structure of telomeric DNA was recently reported in another study, in which stabilization was brought about by mutation and resultant additional interactions [Luu KN, Phan AT, Kuryavyi V, Lacroix L & Patel DJ (2006) Structure of the human telomere in K(+) solution: an intramolecular (3+1) G-quadruplex scaffold. J Am Chem Soc 128, 9963-9970]. The structure of the guanine tracts was similar between the two. However, a difference was seen for loops connecting guanine tracts, which may play a role in the higher order arrangement of telomeres. Our structure can be utilized to design a small molecule which stabilizes the quadruplex. This type of molecule is supposed to inhibit a telomerase and thus is expected to be a candidate anticancer drug.     

Adrenomedullin: a possible regulator of germinal vesicle breakdown

Adrenomedullin (ADM) is a multifunctional hormone that regulates processes as diverse as blood pressure and cell growth. Although expressed in the ovary, the role of ADM in this organ is not clear. In the present study, we found the expression of ADM receptor and receptor activity-modifying proteins in mouse cumulus cells but not in the oocytes. We report that germinal vesicle breakdown (GVBD), which is required for oocyte maturation, is not inhibited by ADM alone. However, ADM in the presence of the nitric oxide donor sodium nitroprusside (SNP) significantly inhibited GVBD. Furthermore, the ADM- and SNP-dependent inhibition of GVBD was abrogated by Akt blockade. Additionally, Akt expression and phosphorylation was exhibited by ADM, suggesting that Akt signaling upstream in cumulus cells is responsible. Additionally, immunohistochemical analysis revealed that ADM was localized in the granulosa cells of developed follicles, implying the possibility that ADM physiologically affects oocyte maturation in vivo. Our results provide the evidence that ADM can act as a GVBD regulator.     

Direct uptake and degradation of DNA by lysosomes

Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered “RNautophagy,” an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term “DNautophagy,” is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa.     

LOV KELCH PROTEIN2 and ZEITLUPE repress Arabidopsis photoperiodic flowering under non-inductive conditions, dependent on FLAVIN-BINDING KELCH REPEAT F-BOX1

LOV KELCH PROTEIN2 (LKP2), ZEITLUPE (ZTL)/LOV KELCH PROTEIN1 (LKP1) and FLAVIN‐BINDING KELCH REPEAT F‐BOX1 (FKF1) constitute a family of Arabidopsis F‐box proteins that regulate the circadian clock. Over‐expression of LKP2 or ZTL causes arrhythmicity of multiple clock outputs under constant light and in constant darkness. Here, we show the significance of LKP2 and ZTL in the photoperiodic control of flowering time in Arabidopsis. In plants over‐expressing LKP2, CO and FT expression was down‐regulated under long‐day conditions. LKP2 and ZTL physically interacted with FKF1, which was recruited from the nucleus into cytosolic speckles. LKP2 and ZTL inhibited the interaction of FKF1 with CYCLING DOF FACTOR 1, a ubiquitination substrate for FKF1 that is localized in the nucleus. The Kelch repeat regions of LKP2 and ZTL were sufficient for their physical interaction with FKF1 and translocation of FKF1 to the cytoplasm. Over‐expression of LKP2 Kelch repeats induced late flowering under long‐day conditions. lkp2 ztl double mutant plants flowered earlier than wild‐type plants under short‐day (non‐inductive) conditions, and both CO and FT expression levels were up‐regulated in the double mutant plants. The early flowering of lkp2 ztl was dependent on FKF1. LKP2, ZTL or both affected the accumulation of FKF1 protein during the early light period. These results indicate that an important role of LKP2 and ZTL in the photoperiodic pathway is repression of flowering under non‐inductive conditions, and this is dependent on FKF1.     

Attenuated thermoregulatory responses with increased plasma osmolality in obese subjects during two seasons

Obese subjects may be more vulnerable to injury from heat stress, and appear to be less efficient at thermoregulation. Sweat rate, tympanic temperature and osmolality in obese subjects were investigated in Japan during two seasons. The purpose of this study was to examine the relationship between obesity, thermoregulatory response and season. Five obese (BMI, 32.0?±?4.9 kg/m²) and five non-obese (BMI, 23.2?±?2.9 kg/m²) men participated in this experiment at latitude 35°10′ N and longitude 136°57.9′E. The average atmospheric temperature was 29.1?±?1.0 °C in summer and 3.3?±?1.4 °C in winter. Tympanic temperature and sweat rate were measured during leg water immersion at 42 °C for 30 min. Blood samples were analyzed for plasma osmolality. The relationship between tympanic temperature and sweat rate decreased significantly in obese compared to in non-obese subjects in both seasons, there being a lowered sweat rate for any core temperature in obese subjects. Plasma osmolality was significantly higher in obese than in non-obese subjects in both seasons. Thermal sensation increased significantly in non-obese than in obese in winter but not in summer. Our data show that thermoregulatory responses are attenuated in obese subjects compared with controls, suggesting that obese people are at increased risk of heat-related illnesses.     

Hydroxylation of methylated DNA by TET1 in chondrocyte differentiation of C3H10T1/2 cells

DNA methylation is closely involved in the regulation of cellular differentiation, including chondrogenic differentiation of mesenchymal stem cells. Recent studies showed that Ten–eleven translocation (TET) family proteins converted 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, 5-formylcytosine and 5carboxylcytosine by oxidation. These reactions constitute potential mechanisms for active demethylation of methylated DNA. However, the relationship between the DNA methylation patterns and the effects of TET family proteins in chondrocyte differentiation is still unclear. In this study, we showed that DNA hydroxylation of 5mC was increased during chondrocytic differentiation of C3H10T1/2 cells and that the expression of Tet1 was particularly enhanced. Moreover, knockdown experiments revealed that the downregulation of Tet1 expression caused decreases in chondrogenesis markers such as type 2 and type 10 collagens. Furthermore, we found that TET proteins had a site preference for hydroxylation of 5mC on the Insulin-like growth factor 1 (Igf1) promoter in chondrocytes. Taken together, we showed that the expression of Tet1 was specifically facilitated in chondrocyte differentiation and Tet1 can regulate chondrocyte marker gene expression presumably through its hydroxylation activity for DNA.     

Development of LC-MS/MS analysis of cyclic dipeptides and its application to tea extract

2,5-Diketopiperazines (DKPs), also called cyclic dipeptides, have been known to occur in various foods. Recently, DKPs have attracted attentions as bioactive components. There were some reports on analytical methods for DKPs, but the number of analyzed DKPs was only a part of all DKPs and the quantitative performance was not studied in detail. In this study, we selected 31 kinds of DKPs and developed a quantitative and simultaneous analytical method using LC-MS/MS. This method was applied to DKPs determination in Pu-erh tea, post-fermentation tea, and 18 kinds of DKPs were determined at concentration of 0.0017–0.11 ppm. As a result of spiked test, it was concluded that the developed method using LC-MS/MS was useful for estimating DKPs concentration in tea.